CHAPTER XL 125 



Chemical combination obviously occurs in some cases when 

 substances stained by adsorption are heated to 100 C. Thus 

 BAYLISS (Proc. Roy. Soc., B., vol. Ixxxiv, 1911, p. 83) showed that 

 various insoluble hydroxides, such as that of aluminium, are stained 

 blue by the free acid of Congo red, which is its own colour. When 

 heated to 100 C., combination takes place with the formation of 

 the usual red colour of the salts of this dye. Silk behaves in the same 



/ 



way, and even crystals of leucine, doubtless to be explained by the 

 formation of salts with the fairly strong acid. 



Conditions similar to this, however, do not arise in histological 

 staining, where dye salts are used. It is difficult, moreover, to see 

 how the conditions for reaction by double decomposition could arise 

 in the cell, since acids and bases sufficiently strong to displace 

 hydrochloric acid or sodium hydroxide are required. Reactions 

 with precipitation might occur, but these would give rise to the 

 appearance of new solid structures in the cell. There is no evidence 

 that such precipitates are produced in a simple staining process, 

 although they are undoubtedly formed by fixing agents (HARDY, 

 Journ. of Physiology, vol. xxiv, p. 158). 



The following experiment by MARTIN HEIDENHAIN is sometimes 

 given as evidence of the formation of salts of dyes with proteins. A 

 solution of Congo red, as is well known, turns blue when made acid 

 with acetic acid, owing to the separation of the free acid. If such 

 a blue solution is added to an acidulated solution of serum albumin, 

 a red solution is obtained. Since this is the characteristic colour of 

 the salts of Congo red, it is natural to interpret it as a salt of the dye 

 acid with the protein base. But the fact that it exists in a solution 

 sufficiently acid to decompose the sodium salt of the dye shows that 

 the colour acid is more firmly combined with protein than with 

 sodium, a view that it is difficult or impossible to hold. Moreover, 

 it appears that even 5 per cent, sulphuric acid is unable to split off 

 the acid from some of these protein " compounds.-' It is clear that 

 the phenomena must have a different interpretation. It may be 

 that the free colour acid exists in two forms, a true and a pseudo- 

 acid the former of a red colour and ionised ; the latter, blue, in- 

 soluble and non-ionised. When adsorbed by protein, for some reason 

 or other, the acid may be for the most part in the former condition. 

 See the work of Wo. OSTWALD on Congo-rubin (.fiM. Chem. Beihefte, 

 B. x, 1919). But further evidence is required. 



203. Removal of Dyes. When a stain is fixed by adsorption of 

 the ordinary, non-electrical type, it can be removed by frequent 

 washing with distilled water. This is very difficult if the dye is held 



